The 283 amino acid voltage dependent anion channel of the mitochondrial outer membrane (VDAC or porin) has been found to be a member of a human gene family of at least three proteins. The two recently discovered VDACs contain an amino acid segment that has ion channel properties similar to the mitochondrial VDAC; however, they are most prominently distinguished by N-terminal and C-terminal sequences that extend beyond this 283 residue segment. Terminal extensions of this type suggest differences in the intracellular sorting of the three VDACs. This argument is supported by the fact that VDACs have been identified in the plasmalemma of lymphocytes and have been reported to be a component of the purified GABAA receptor complex. Except for ongoing work in this laboratory that has identified two lysyl residues that are crucial for the membrane assembly of the mitochondrial VDAC, little is known about the molecular factors that determine the sorting and membrane insertion of the VDAC family. However, it is clear that a failure in either of these processes could lead to severe medical problems including emotional and neurological dysfunctions. In this proposal, molecular and cellular biological approaches are described that will identify regions of the VDACs that determine their subcellular distribution. Extension, deletion and internal point mutants of the VDACs will be used to identify terminal tracts and internal clusters of amino acids that serve in the sorting and insertion of the VDACs into the mitochondria or into the endoplasmic reticulum and subsequently to other non-mitochondrial membranes. Although some in vivo experiments with yeast will be performed, membrane sorting and insertion will be assayed primarily in vitro using membranes derived from yeast and mammalian sources.